Antibiotic LL-D42067α

ABSTRACT

Antibiotic LL-D42067α derived by aerobic fermentation of the microorganism Actinomadura madurae subspecies simaoensis NRRL 15734, useful as an antibacterial and antiparasitic agent.

SUMMARY OF THE INVENTION

This invention relates to a new antibacterial and antiparasitic agentdesignated LL-D42067α, to its production by fermentation, to methods forits recovery and concentration from crude solutions and to processes forits purification. The present invention includes within its scope thebiologically pure culture of the antibiotic.

The structure and relative stereochemistry of LL-D42067α have beenelucidated by X-ray crystallography, and is shown below. ##STR1##

The physico-chemical characteristics of LL-D42067α are described below:

(1) Molecular weight: 535 (FAB-MS);

(2) Molecular formula: C₂₈ H₂₅ NO₁₀ ;

(3) Specific optical rotation: [α]_(D) ²⁶ =+836³⁰ -40° (C 0.3, DMF);

(4) Ultraviolet absorption spectra: as shown in FIG. I

UV_(MAX) ^(CH).sbsp.3^(OH) =215 nm (ε 13,200); 254 nm (ε 15,000); 320 nm(ε 5,100); 395 nm (ε 11,400);

UV_(MAX) ⁰.1 N HCl =213 nm (ε 27,100); 253 nm (ε 34,500); 321 nm (ε12,200); 374 nm (ε 21,100); 389 nm (ε 22,900);

UV_(MAX) ⁰.1 N NaOH =217 nm (ε 42,100); 253 nm (ε 13,900); 312 nm (ε5,700); 395 nm (ε 10,700);

(5) Infrared absorption spectrum: as shown in FIG. II (KBr disc): 1650,1598, 1543, 1470, 1440, 1260, 1195, 1020 cm⁻¹ ;

(6) Proton nuclear magnetic resonance spectrum (CDCl₃): as shown in FIG.III, and described in Table I;

(7) Carbon-13 nuclear magnetic resonance spectrum (DMSO): as shown inFIG. IV and described in Table II; and

(8) Proton to carbon-13 chemical shift correlation map (DMSO): as shownin FIG. V.

                  TABLE I                                                         ______________________________________                                        Proton NMR Data for LL-D42067α                                          δ*                                                                            No. of Hydrogen**                                                                            Multiplicity***                                                                            J (H)                                       ______________________________________                                        1.88  2              m                                                        2.34  2              m                                                        2.45  3              s                                                        2.58  1              m                                                        3.62  3              s                                                        3.72  1              d,d          4.64, 14.22                                 3.88  3              s                                                        4.80  2              m                                                        5.08  1              m                                                        5.32  1              d            5.81                                        5.55  1              d            5.81                                        6.70  1              s                                                        12.76 1              s                                                        13.58 1              s                                                        ______________________________________                                         *CDCl.sub.3, ppm downfield from TMS.                                          **Spectrum in DMSOd.sub.6, shows two additional absorptions at 4.55(s) an     5.91 (d) ppm.                                                                 ***s = singlet; d = doublet; t = triplet; m = multiplet.                 

                  TABLE II                                                        ______________________________________                                        Carbon-13 NMR Data for LL-D42067α                                       Carbon     Chemical Shift (ppm)*                                                                        Carbon Type                                         ______________________________________                                         1          20.4          CH.sub.3                                             2          25.4          CH.sub.2                                             3          25.8          CH.sub.2                                             4          29.0          CH.sub.2                                             5          30.4          CH.sub.3                                             6          58.5          CH.sub.                                              7          61.6          CH.sub.3                                             8          63.3          CH.sub.                                              9          71.7          CH.sub.                                             10          90.4          CH.sub.2                                            11         100.0          CH.sub.                                             12         109.2           q**                                                13         109.7          q                                                   14         111.0          q                                                   15         113.7          q                                                   16         119.0          q                                                   17         125.8          q                                                   18         126.6          q                                                   19         134.9          q                                                   20         135.3          q                                                   21         136.1          q                                                   22         141.3          q                                                   23         147.9          q                                                   24         151.1          q                                                   25         152.5          q                                                   26         165.4          q                                                   27         165.6          q                                                   28         182.3          q                                                   ______________________________________                                         *DMSO-d.sub.6, ppm downfield from TMS.                                        **q = quarternary.                                                       

DETAILED DESCRIPTION OF THE INVENTION

The new antibacterial agent designated LL-D42067α, is formed during thecultivation under controlled conditions of a new strain of a newsubspecies of Actinomadura madurae. This new strain is maintained in theculture collection of the Medical Research Division, American CyanamidCompany, Pearl River, N.Y. as culture number LL-D42067. A viable cultureof this new microorganism has been deposited with the Patent CultureCollection Laboratory, Northern Regional Research Center, U.S.Department of Agriculture, Peoria, Ill. 61604, and has been added to itspermanent collection. It is freely available to the public in thisdepository under its accession number NRRL 15734.

Culture LL-D42067 was isolated from a soil sample from San Simao,Brazil. The culture was taxonomically characterized and was identifiedas a new subspecies of Actinomadura madurae, designated Actinomaduramadurae subspecies simaoensis.

Observations were made of the cultural, physiological and morphologicalfeatures of the culture in accordance with the methods detailed byShirling and Gottlieb [Intern. J. System. Bacteriol., 16:313-340 (1966)]and Gordon, et al. [Intern. J. System. Bacteriol., 24:54-63 (1974)]. Thechemical composition of the cell walls of the culture was determinedusing the method of Lechevalier, et al. [Adv. Appl. Microbiol., 14:47-72(1971)]. Details are recorded in Tables III-V, and a general descriptionof the culture is given below. Underscored descriptive colors are takenfrom Kelly and Judd [Nat. Bur. Stand., Spec. Publ., 440 (1976)] and theaccompanying Intersociety Color Council, National Bureau of StandardsCentroid Color Charts.

GROWTH CHARACTERISTICS

Table III describes the cultural characteristics of culture LL-D42067 onvarious agar media which were selected from those recommended by theInternational Streptomyces Project Committee (hereinafter referred to as"ISP").

MICROMORPHOLOGY

Microscopic examination of the strain showed it to form short chains ofconidia on aerial hyphae which were slightly hooked to short-spirals (upto three turns). The spore surfaces were smooth when observed byelectron microscopy, distinguishing this isolate from A. verrucosopora.

CELL WALL COMPOSITION

Whole cell analyses showed the strain to contain meso diaminopimelicacid (DAP) and the sugar 3-O-methyl-D-galactose (madurose); thus itfalls into whole cell pattern type B. The cell wall composition was ofthe type III (meso DAP, glutamic acid, alanine, muramic acid andglucosamine) and the phospholipid pattern of type PIV (phosphatidylethanolamine and/or methylethanolamine plus unknownglucosamine-containing phospholipids). These data support the assignmentof the strain to the genus Actinomadura. The PIV phospholipid type isnot typical for A. madurae, which is usually PI.

PHYSIOLOGICAL REACTIONS

The physiological reactions of strain LL-D42067 were examined using boththe ISP system, Shirling and Gottlieb [Intern. J. Syst. Bacteriol.,16:313-340 (1966)] and the Gordon tests, Gordon, et al. [Intern. J.Syst. Bacteriol., 24:54-63 (1974)]. The utilization pattern of thestrain of ISP carbohydrate media is given in Table IV, along with thoseof other members of the genus reacting similarly. Culture LL-D42067resembles the Actinomadura madurae and Actinomadura verrucosoporagroups. As indicated above, however, it differs from Actinomaduraverrucosopora in having smooth spore walls. A comparison of reactions inthe Gordon test series of Actinomadura madurae (Gordon's data; seereference above) and LL-D42067, summarized in Table V, revealeddifferences only in amylase production and acid from glycerol andraffinose. Since amylase production and raffinose utilization have beenfound to be variable in Actinomadura madurae [Goodfellow, N., et al., J.Gen. Microbiol., 112:95-111 (1979)], the glycerol reaction remains theonly physiological difference of LL-D42067 from this taxon.

Since strain LL-D42067 is the same as Actinomadura madurae in allproperties evaluated except for its glycerol reaction and its PIVphospholipid pattern, it has been assigned to the taxon Actinomaduramadurae as a subspecies designated Actinomadura madurae subspeciessimaoensis.

                  TABLE III                                                       ______________________________________                                        Cultural Characteristics of LL-D42067 Actinomadura                            madurae subspecies simaoensis on ISP Morphological Media                                  Aerial     Vegetative   Soluble                                   Agar Medium Mycelium   Mycelium     Pigment                                   ______________________________________                                        Yeast extract, Malt                                                                       White, sparse                                                                            Medium orange-                                                                             None                                      extract (ISP 2)        brown-I53*                                             Inorganic Salts                                                                           Colorless  Colorless    None                                      Starch (ISP 4)                                                                Glucose Asparagine                                                                        Colorless  Colorless    None                                      (ISP 5)                                                                       Oatmeal (ISP 3)                                                                           Sparse     Light orange-                                                                              None                                                  pinkish-white                                                                            brown-I52*                                             ______________________________________                                         *I = ISCC Color charts                                                   

                  TABLE IV                                                        ______________________________________                                        Comparison of Carbohydrate Utilization Reactions of                           LL-D42067 With Related Actinomadura spp.                                                           A. madurae                                                                              A. verrucosopora                               Carbohydrate                                                                           LL-D42067   (a)       (a) (b)                                        ______________________________________                                        L-arabinose                                                                            +           +         +                                              D-fructose                                                                             +           +         +                                              I-inositol                                                                             -           variable  variable                                       D-mannitol                                                                             +           +         +                                              raffinose                                                                              -           -         -                                              rhamnose +           +         +                                              sucrose  +           +         +                                              D-xylose +           +         +                                              ______________________________________                                         (a) Goodfellow, M., et al., J. Gen. Microbiol., 112:95-111 (1979).            (b) Nonomura, H. and O'Hara, Y., J. Ferm. Technol., 49:904-912 (1971).   

                  TABLE V                                                         ______________________________________                                        Gordon Test Reactions of LL-D42067                                                                    A. madurae                                                            LL-D42067                                                                             (Gordon Data*)                                        ______________________________________                                        Degradation/Transformation of                                                 Casein            +         +(98)                                             Xanthine          -         -                                                 Hypoxanthine      +         +(98)                                             Tyrosine          +         +(91)                                             Adenine           -         -                                                 Production of                                                                 Amylase           -         +                                                 Gelatinase        +         +                                                 Phosphatase       -         ND                                                Nitrate Reductase +         +(98)                                             Urease            -         -                                                 Esculinase        +         +(98)                                             Growth on/in                                                                  5% Sodium Chloride                                                                              -         ND                                                Salicylate        -         ND                                                Lysozyme Broth    -         -(91)                                             Utilization                                                                   Acetate           +         +                                                 Benzoate          -         -(94)                                             Citrate           -         +(83)                                             Lactate           +         ND                                                Malate            +         +(84)                                             Mucate            -         -                                                 Oxalate           -         ND                                                Propionate        -         ND                                                Pyruvate          +         ND                                                Succinate         +         +(83)                                             Tartrate          -         -                                                 Growth at                                                                     10° C.     -         -                                                 45° C.     +         -(66)                                             53° C.     -         -                                                 Acid from                                                                     Adonitol          +         +(91)                                             Arabinose         +         +                                                 Cellobiose        +         +                                                 Dextrin           +         ND                                                Dulcitol          -         -                                                 Erythritol        -         -                                                 Fructose          +         ND                                                Galactose         +         +(84)                                             Glucose           +         +                                                 Glycerol          -         +                                                 Inositol          -         +(60)                                             Lactose           -         +(55)                                             Maltose           -         +(53)                                             Mannitol          +         +                                                 Mannose           +         +(94)                                             Melibiose         -         -                                                 α-Methyl-D-glucoside                                                                      -         -                                                 Raffinose         variable  -                                                 Rhamnose          +         +                                                 Salicin           +         ND                                                Sorbitol          -         -                                                 Sucrose           +         ND                                                Trehalose         +         +(96)                                             Xylose            +         +                                                 β-Methyl-D-xyloside                                                                        +         ND                                                ______________________________________                                         *Percentages of cultures showing reaction given in parentheses if not         100%.                                                                         ND = Not determined.                                                     

For the production of this new antibacterial and antiparasitic agent thepresent invention is not limited to this particular organism or toorganisms fully answering the above growth and microscopiccharacteristics, which are given for illustrative purposes only. Infact, it is desired and intended to include the use ofnaturally-occurring mutants of this organism as well as induced mutantsproduced from this organism by various mutagenic means known to thoseskilled in the art such as exposure to nitrogen mustard, X-rayradiation, ultraviolet radiation, N'-methyl-N'-nitro-N-nitrosoguanidine,actinophages and the like. It is also desired and intended to includeinter- and intraspecific genetic recombinants produced by genetictechniques known to those skilled in the art such as, for example,conjugation, transduction and genetic engineering techniques.

The in vitro antimicrobial spectrum of LL-D42067α was determined by theagar plate dilution method with Mueller-Hinton agar and an inoculum ofeach test organism of approximately 10⁴ colony forming units deliveredby the Steers replicating device. The minimal inhibitory concentration(MIC) in mcg/ml was defined as the lowest concentration of LL-D42067αthat inhibited visible growth after 18 hours incubation at 35° C.

The results, summarized in Table VI, show that LL-D42067α was activeversus gram-positive bacteria and moderately active against yeasts.

                  TABLE VI                                                        ______________________________________                                        Antimicrobial Spectrum of LL-D42067α                                    Test Organism            MIC (mcg/ml)                                         ______________________________________                                        Staphylococcus aureus                                                                           Smith      ≦0.06                                     Staphylococcus aureus                                                                           LL #14     ≦0.06                                     Staphylococcus aureus                                                                           LL #27     ≦0.06                                     Staphylococcus aureus                                                                           LL #45     ≦0.06                                     Staphylococcus aureus                                                                           ATCC 25923 ≦0.06                                     Staphylococcus epidermidis                                                                      CMC-83-56  ≦0.06                                     Staphylococcus epidermidis                                                                      ATCC 12228 ≦0.06                                     Streptococcus faecalis                                                                          ATCC 29212 ≦0.06                                     Streptococcus (enterococcus sp)                                                                 OSU-75-1   ≦0.06                                     Streptococcus (enterococcus sp)                                                                 SM-77-15   ≦0.06                                     Streptococcus mutans                                                                            ATCC 27352 ≦0.06                                     Streptococcus mutans                                                                            BHI (b)    ≦0.06                                     Streptococcus sanguis                                                                           G9B (a)    ≦0.06                                     Micrococcus luteus                                                                              PC 1001    ≦0.06                                     Bacillus subtilis ATCC 6633  ≦0.06                                     Bacillus cereus   LL #4      ≦0.06                                     Candida albicans  CA 300     256                                              Saccharomyces cerevisiae                                                                        Y 15       32                                               Escherichia coli  #311       512                                              Escherichia coli  ATCC 25922 512                                              Klebsiella pneumoniae                                                                           AD         512                                              Proteus morganii  K-79-25    512                                              Acinetobacter calcoaceticus                                                                     Stfd-79-17 512                                              ______________________________________                                    

The antibiotic LL-D42067α derives utility from its antibacterial andantiparasitic activities. For example, the antibiotic may be used in thesuppression of intestinal bacterial flora, as a topical antibacterialagent or antiseptic against gram-positive bacteria and as a generaldisinfectant for surfaces such as instruments. It may also be useful asan antiprotozoal agent in the treatment of malaria. In addition to itsantimicrobial and antiparasitic activity LL-D42067α is effective as ananticoccidial agent in poultry. This utility is the subject of acopending application for United States Letters Patent.

In therapeutic use, the compound of this invention may be administeredin the form of conventional pharmaceutical compositions appropriate forthe intended use. Such compositions may be formulated so as to besuitable for oral or topical administration. The active ingredient maybe combined in admixture with a nontoxic pharmaceutically acceptablecarrier, which carrier may take a wide variety of forms depending on theform of preparation desired for administration, i.e., oral or topical.

GENERAL FERMENTATION CONDITIONS

Cultivation of Actinomadura madurae subspecies simaoensis NRRL 15734 maybe carried out in a wide variety of liquid culture media. Media whichare useful for the production of this novel antibiotic LL-D42067αinclude an assimilable source of carbon, such as dextrin, sucrose,molasses, glycerol, etc.; an assimilable source of nitrogen such asprotein, protein hydrolysate, polypeptides, amino acids, corn steepliquor, etc.; and inorganic anions and cations, such as potassium,sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, etc.Trace elements such as boron, molybdenum, copper, etc., are supplied asimpurities of other constituents of the media. Aeration in tanks andbottles is supplied by forcing sterile air through or onto the surfaceof the fermenting medium. Further agitation in tanks is provided by amechanical impeller. An antifoam agent such as silicone oil may be addedas needed.

GENERAL PROCEDURE FOR THE ISOLATION OF LL-D42067α

The LL-D42067α antibiotic is recovered from the fermentation broth byfiltration through diatomaceous earth, extracted into a solvent such asmethylene chloride and purified by column chromatography on silica gel,using the system hexane:ethyl acetate (80:20) to remove unwanted fatsand then methylene chloride:1% acetic acid in methanol (9:1) to give acrude product.

This crude LL-D42067α is then purified by high performance liquidchromatography on a reverse phase column using the systemacetonitrile:water:acetic acid (600:400:0.28).

EXAMPLE 1 Inoculum Preparation

A typical medium used to grow the primary inoculum was preparedaccording to the following formula:

    ______________________________________                                        Glucose           1.0%                                                        Dextrin           2.0%                                                        Yeast extract     0.5%                                                        N-Z Amine A ®.sup.1                                                                         0.5%                                                        Calcium carbonate 0.1%                                                        Water qs          100%                                                        ______________________________________                                         [.sup.1 A pancreatic digest of casein, registered trademark of Sheffield      Chemical, Norwich, New York                                              

This medium was adjusted to pH 7.2 and then sterilized. A 100 ml portionof this sterile medium, in a 500 ml flask, was inoculated with mycelialscrapings from an agar slant of Actinomadura madurae subspeciessimaoensis NRRL 15734. The medium was then placed on a rotary shaker andagitated vigorously at 210 rpm for 48-72 hours at 28° C. This primaryinoculum was then used to inoculate 12 liters of the same sterile mediumwhich was then grown at 28° C. for 48 hours providing secondaryinoculum.

EXAMPLE 2

A fermentation medium of the following formulation was prepared:

    ______________________________________                                        Sucrose           3.0%                                                        Soy flour         1.5%                                                        Corn steep liquor 0.5%                                                        Calcium carbonate 0.5%                                                        Water qs          100%                                                        ______________________________________                                    

The medium was sterilized and inoculated at the rate of 12 liters ofsecondary inoculum from Example 1 per 300 liters of medium. Thefermentation was conducted at 28° C. with a sterile air flow of 200liters per liter of mash per minute, agitation by an impeller operatedat 230 rpm for 135-159 hours at which time the mash was harvested andfiltered through diatomaceous earth.

EXAMPLE 3 Isolation of LL-D42067α

The fermentation filtrate from three fermentations, conducted asdescribed in Example 2, were combined, making a total of 1800 liters atpH 7.5, and extracted with 900 liters of methylene chloride. The organicphase was concentrated in vacuo to give 84.1 g of residue.

A 75.2 g portion of this residue was suspended in 300 ml of hexane:ethylacetate (80:20) and allowed to seep into a glass column (2 inches×20inches) dry packed with silica gel. The column was eluted with a totalof 4 liters of the same solvent mixture in order to remove fats andsilicone oil and was then eluted with 4 liters of methylene chloride:1%acetic acid in methanol (9:1) collecting 15 ml fractions. The fractionswere analyzed by thin-layer chromatography. Antibiotic LL-D42067αappeared visually as a yellow spot (Rf=0.5) with the same solventsystem. Fractions 31-60, which contained most of the antibiotic, werepooled and concentrated in vacuo, giving 11.1 g of a red residue.

A 5.5 g portion of the above residue was fractionated by highperformance liquid chromatography [Prep LC System-500, Prep PAK-500/C18cartridge, acetonitrile:water:acetic acid (600:400:0.28), 100 ml/minute,5.5 g/30 ml/injection]. Thirty 200 ml fractions were collected.Analytical high performance liquid chromatographic analysis of thefractions showed the major portion of LL-D42067α was in fraction 5.Fraction 5 was allowed to stand overnight. The resulting yellow crystalswere collected by decanting off the mother liquor (which was saved),washing the crystals with the mobile phase and air drying, giving 11 mgof LL-D42067α as yellow crystals.

The mother liquor was concentrated by slow evaporation. The resultingprecipitate was collected by centrifugation giving 377 mg of LL-D42067αas a yellow amorphous solid.

The analytical HPLC conditions were:

    ______________________________________                                        Column:       μ Bondapak C18, 3.9 mm × 30 cm,                                      Waters Associates                                               Mobile Phase: acetonitrile:water:acetic acid                                                (400:600:0.28)                                                  Detector:     UV 254 nm and UV 365 nm, 0.2 AUFS                               Flow Rate:    1.0 ml/minute                                                   Retention Volume of                                                                         11.5 ml.                                                        LL-D42067α:                                                             ______________________________________                                    

We claim:
 1. The compound designated as LL-D42067α, wherein the compoundhas:(a) the relative stereochemistry and structure: ##STR2## (b) amolecular weight of 535 (FAB-MS); (c) a molecular formula: C₂₈ H₂₅ NO₁₀; (d) a specific optical rotation: [α]_(D) ²⁶ =+836⁺ -40° (C 0.3, DMF);(e) characteristic ultraviolet absorption spectra as shown in Figure Iof the attached drawings; (f) a characteristic infrared absorptionspectrum as shown in FIG. II of the attached drawings; (g) acharacteristic proton nuclear magnetic resonance spectrum as shown inFIG. III of the attached drawings; (h) a characteristic carbon-13nuclear magnetic resonance spectrum as shown in FIG. IV of the attacheddrawings with significant peaks at: 20.4; 25.4; 25.8; 29.0; 30.4; 58.5;61.6; 63.3; 71.7; 90.4; 100.0; 109.2; 109.7; 111.0; 113.7; 119.0; 125.8;126.6; 134.9; 135.3; 136.1; 141.3; 147.9; 151.1; 152.5; 165.4; 165.6;182.3; and (i) a characteristic proton to carbon-13 chemical shiftcorrelation as shown in Figure V of the attached drawings.